Atherothrombotic vascular disease, the cause of heart attacks, strokes, and sudden death, is the leading cause of death in the industrialized world. These acute vascular events are triggered by disruption of advanced atherosclerotic plaques that have large necrotic areas. Plaque necrosis is caused by macrophage (Mf) death, or apoptosis, in advanced lesions. The PI's laboratory introduced the concept that endoplasmic reticulum (ER) stress in advanced lesional Mfs is an important mechanism of Mf death. ER stress induces a pro-apoptotic effector called CHOP, which conspires with other pro-apoptotic hits to induce Mf death. The overall objective of this proposal is to expand these concepts in three critical areas: (a) discrimination of the roles of ER stress in advanced vs. early lesions and in Mfs vs. endothelial cells; (b) elucidation of the role of increased cytosolic Ca2+, a by-product of ER stress, in the multi-hit model of Mf apoptosis; and (c) assessment of whether decreased advanced lesional Mf apoptosis and plaque necrosis via genetic manipulation of the multi-hit/ER stress pathway has a beneficial effect in plaque disruption. In Aim I, reciprocal bone marrow transplant (BMT) experiments using Chop-/- and Ldlr-/- mice will test the hypothesis that endothelial CHOP promotes early atherogenesis while Mf CHOP promotes advanced atherosclerosis. Aim II will address the hypothesis that elevation of cytosolic calcium induced by ER stress amplifies two important pro-apoptotic pathways of the multi-hit model, one involving toll-like receptor 4 and c-Jun-NH(2)-terminal kinase, and the other involving Ca2+-calmodulin kinase II and serine-phosphorylated STAT1. BMT experiments in Aim III will test the hypothesis that Mf-STAT1 deficiency will decrease Mf death and plaque necrosis in advanced atherosclerotic lesions. The proposed link between plaque necrosis and plaque disruption will be addressed in Aim IV by determining whether plaque disruption in Apoe- /- mice with Mf-targeted expression of activated MMP9 is decreased in the setting of CHOP or combined CHOP/STAT1 deficiency. Through these studies, we hope to conceive novel therapeutic strategies to prevent advanced plaque progression and atherothrombotic vascular disease. Indeed, the Mf apoptosis pathway being studied here has been shown to be amplified in the setting of diabetes, which represents the major driving force predicted to increase atherothrombotic vascular disease in the immediate and longer term future.